We have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 mo) and old (30 mo) FXBN rats. Using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE), we obtained 2-D gel maps of 301 identified non-redundant proteins from rat aortae and observed 18 proteins whose abundance significantly changes with aging. Utilizing Isobaric tags for relative and absolute quantitation (iTRAQ), 921 proteins were quantified and between both methods, 50 proteins were shown to have significantly different age-associated abundance. Proteomic analysis shows that milk fat globule protein EGF-8 (MFG-E8) significantly increases in abundance in old rat aortae. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species, including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within rat vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. AngII treatment of early passage VSMCs, isolated from young aortae, markedly increases MFG-E8 expression and enhances the invasive capacity of VSMCs to levels observed from old. MFG-E8 treatment of VSMCs increased MCP-1, matrix metalloproteinase type II (MMP2) and VSMC invasion capacity but these effects were inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 RNA substantially reduced MFG-E8 expression, MMP2 activation and VSMCs invasion capacity. Thus, arterial MFG-E8 significantly increases with aging and is a key component within the Ang II-MCP-1/MMP2/VSMCs invasion signaling cascade. We have identified that aging arterial MFG-E8-enriched in VSMCs are activated, invaded and proliferated both in vivo and in vitro. Increased MFG-E8 in VSMCs triggers phosphorylation of extracellular signal regulated kinases-1/2(ERK-1/2), augments levels of proliferation cell nuclear antigen (PCNA) and cyclin-dependent kinase 4 (CDK4), increases BrdU incorporation and promotes cell growth. When MFG-E8 is knocked down, the speed of cell cycling is reduced, the expression of the signaling molecules PCNA and CDK4 is accelerated, thus, facilitating the cells entry into a growth-arrested state. Furthermore, we find that integrin v5 and platelet derived growth factor (PDGF) receptor are upregulated with MFG-E8 treatment and are also key elements of extracellular proliferative signals in aging VSMC. Importantly, we found that the involvement of MFG-E8 in atherosclerotic lesions increases with age. In in vivo studies: 1) MFG-E8 protein in the aortic wall of ApoE-/- mice on a standard chow diet increases with age and is localized predominantly in advanced plaques and degraded elastin laminae. 2) In nonhuman primates (9 to 23- years old), the iTRAQ assay indicates that levels of arterial MFG-E8 protein is highly correlated with age. In older monkeys (>17-years old), fed a high cholesterol diet for 2 years, a 1.6-fold increase in the amount of arterial MFG-E8 protein and its fragment, medin, was seen when compared to older animals fed a standard diet. 3) In aortic thoracic intimae isolated from adult humans (age 22-to 69-years old), Western blot analysis indicated that the abundance of aortic MFG-E8 protein and its fragment, medin, significantly increased with advancing age; 4) Immunostaining of atherosclerotic plaques indicates that MFG-E8 is mainly derived from smooth muscle cells (alpha-smooth actin positive) and macrophages (CD 68 positive). In in vitro studies: 1) Early passage thoracic aortic smooth muscle cells (SMC) from both young and old monkeys treated with pathogenic oxidative low-density lipoprotein (oxLDL) showed an increase in the uptake capacity of oxLDL in old cells that exceeded that of young cells, thus facilitating the trans differentiation of SMCs into foam-like cells (CD68 positive). 2) Silencing of MFG-E8 RNA markedly reduced the uptake of oxLDL by young and old monkey VSMCs. Interestingly, the MFG-E8 C-terminal fragment, medin, was reported to be involved in the necrosis of VSMCs at the inflammatory sites of aortic dissection. Immunofluorescence, immunohistochemistry and western blot analysis demonstrate that MFG-E8 and its fragment, medin, Ang II, activated matrix metalloproteinase type II (MMP-2) and platelet derived growth factor receptor type-beta (PDGFR-) protein levels were markedly increased in human grossly normal aortic walls from old (n=10, >50 yrs) vs. younger donors (n=10, <20 yrs). Importantly, an administration of Ang II via an osmotic minipump for 28 days markedly increases MFG-E8 protein and medin precursor in the aortic walls of rats; further, treating primary cultured VSMCs or aortic rings isolated from young and old FXBN rat aortae to a peptide of medin (20 to 100nM) significantly increased MMP-2 activation, PDGFR- expression, and migratory capacity measured with a modified Boyden chamber in a dose-dependent manner. Exposure of activated MMP-2 to recombinant human MFG-E8 protein or to old human aortic wall protein markedly increased the cleavage product medin, respectively and both were substantially inhibited by GM6001, an MMP inhibitor. Treating VSMCs with medin did not significantly affect the expression of cell cycle related proteins, including PCNA, and the proliferation of VSMC as determined by a cell proliferation MTT assay. In addition, platelet-derived growth factor BB (PDGF-BB) treatment markedly activated MMP-2 in both young and old VSMCs, which was substantially reduced by a PDGFR- inhibitor, RTK. Taken together, in vivo and in vitro observations from human donors and rat specimens demonstrate that aging increases aortic ang II, MFG-E8, and medin protein levels, via an MMP-2 cleavage of MFG-E8, promoting VSMCs inflammation and migration, a feature of aged aortic walls. Arterial stiffness, a risk factor for cardiovascular disease that is independent of blood pressure, increases as we age. In addition, there is an increase in aortic wall collagen, and increased arterial stiffness which occurs in the context of elastin fiber fragmentation and elastin structural disorganization in which p38 activation has been implicated. MFG-E8 fragment medin, a proinflammatory molecule secreted from VSMCs, increases in the arterial wall with age and colocalizes with the elastin laminae in stiffened arterial walls. Here, we found that MFG-E8 signaling affects age-associated elastin remodeling. Aging reduces intact tropoelastin expression by 76.7% in aortic tissue and 65.9% in VSMC (both p<0.05). MFG-E8 expression increased with advancing age by 34.5% in aortic tissue and 63.8% in VSMC (both p<0.05). Treatment of VSMCs with MFG-E8 increased p38 activation by 88%, on average, in young and old VSMCs and reduced Tropoelastin expression by 60% in young, and 53% old in VSMCs (both p<0.05). In contrast, siRNA MFG-E8 enhanced tropoelastin expression, 61.3% in young, and 50.7% in old (both p<0.05). When p38 is inhibited after MFG-E8 treatment, Tropoelastin levels are preserved. Treatment with MFG-E8 reduced the contractile SMC phenotype marker protein (SM22) by 23% in young, and by 18.5% old. Expression of SM22 by p38 inhibitor is increased 33.3% in young, and 11.6% in old. The anti- calcification marker protein (Fetuin A) follows a similar pattern under the same culture conditions. Collectively, an age-related increase of MFG-E8 signaling via p38 activation is linked to a reduction in tropoelastin protein levels. This molecular signaling cascade is involved in producing a fertile niche of VSMCs procalcification. Targeting MFG-E8 therefore merits consideration as a novel therapeutic approach to retard age-associated elastin degradation, calcification, and stiffening that increase exponentially with advancing age.